1. Field of the Invention
The present invention relates to measurements on biological materials, which require a high throughput. Specifically, the present invention relates to a method and system for analyzing DNA-polymorphisms in the human genome, such as single nucleotide polymorphisms (SNPs) that are single-base variations at a unique physical location among different individuals.
2. Description of the Prior Art
It is thought that there are about 103 to 105 of SNPs portions in the human genome. For facilitating genomic drug discovery and developing personalized medicines, there is a need to analyze those SNPs portions for many individuals. Accordingly, a high throughput of the analysis of genomic DNA has been required in the field of the related art.
A matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOFMS) has been known in the art. It is described in publications, such as Little, D. P. et al., Analytical Chemistry, pages 4540-4546, no. 22, vol. 69, 1997. In this document, such a mass spectrometer performs analyses on genome DNA samples by applying each sample on a silicon substrate, placing the substrate in a vacuum apparatus, and detecting a mass spectrum for univalent or divalent ions.
In this mass spectrometer, there are tendencies to decrease the sensitivity of the detection thereof with respect to any ion with 10,000 or more of mass-to-charge ratio (m/z: the mass of an ion per the number of electric charges of the ion). Therefore, the mass spectrometer may be used for the analysis on a short-stranded DNA fragment with a base length of 30 bases or less.
The above measurement apparatus (MALDI-TOFMS) carries out a measurement on a DNA sample being applied on the silicon substrate and then placed in a vacuum apparatus. Alternatively, there is another apparatus that allows a measurement on a genome DNA sample being kept in a liquid state. Such an apparatus is described in the document of Krahmer, M. T. et al., Analytical Chemistry, pages 2893 to 2900, no. 14, vol. 71, 1999. In this measurement apparatus, a sample of genome DNA with several tens of bases in a liquid state is ionized by means of electrospray ionization. The mass of genome DNA is then detected by means of mass spectrography. In this case, the results are characterized in that a plurality of multiply-charged ions with 10 or more charges is mainly detected within the limits of m/z=700 to 1,400 in contrast to those obtained by the MALDI-TOFMS.
In the document of Rapid Communication in Mass Spectrometry, pages 214 to 317, vol. 5, 1991, there is described an example in which an electrospray ionization method is applied for performing the analysis on multiply-charged ions of a protein molecule. In this measurement apparatus, a mass spectrum with several peaks is caused by respective multiply-charged ions with 6 to 13 charges. The mass of protein provided as a sample to be measured can be estimated from the mass-to-charge ratio (m/z) that corresponds to the respective peak positions of the mass spectrum.
In this case, a sample being kept in a liquid state may be introduced into the measurement apparatus using the electrospray ionization method at the rate of about 1 to 7 micro-litters per minute.
In the conventional technology using the MALDI-TOFMS described above, there are some problems that need to be addressed. For example, the inside of the vacuum apparatus tends to be contaminated because the conventional process using the above MALDI-TOFMS subjects the sample to a sputtering step using a laser beam irradiation in the vacuum apparatus. If the inside of the vacuum apparatus becomes contaminated, it decreases the efficiency of converging ions to be generated. As a result, there are problems with respect to degradation in sensitivity, reproducibility, or the like. For reducing the possibility of causing any harmful influence of the contamination, there is a need to perform a complicated cleaning repeatedly at frequent intervals. In this case, however, the frequent cleaning may interrupt the continuous measurement for many hours. As a result, and thus the average throughput thereof is decreased. In the case of the MALDI-TOFMS, furthermore, ions to be detected by the mass spectrum are of only two types, monovalent and divalent, so that there is another problem that a need for re-measurement when none of two types cannot be detected.
On the other hand, another conventional technology depending on the electrospray ionization method uses a syringe pump, where multiply-charged ions are generated by performing electrospray ionization, followed by the measurement. In this case, it is possible to introduce a sample into the syringe pump continuously, but only one kind of the sample can be subjected to the measurement. If different kinds of samples are introduced into the same syringe pump for the measurement, the remainder of one sample in the syringe affects on the measurement of another sample. Therefore, it causes the lower measurement accuracy. In addition, the replacement of the syringe pump with another one is a time-consuming job, so that each of successive measurements on many samples with high throughputs requires a great deal of time and much effort.
As described above, therefore, demands for a high throughput have not been satisfied by the conventional genome DNA analysis system.
It is an object of the present invention is to provide a method for the analysis of DNA polymorphism, which allows a long-duration measurement with a high throughput and achieves a stable measurement even though the concentration of a sample is low. It is another object of the present invention is to provide a system for DNA analysis, which allows a long-duration measurement with high throughput and achieves a stable measurement even though the concentration of a sample is low.
For solving the problems associated with the conventional technologies described above, a novel DNA analysis system comprises an ionization part that employs an air atomization method such as an electrospray ionization or sonic spray ionization for allowing a continuous introduction of samples into the ionization part, to attain a high-through put measurement. If the samples are successively introduced into the ionization part, it is difficult to neglect an adsorption of the sample on a sample capillary, an ion source, or the like. Any conditioning step may be performed to avoid the undesired influences of the adsorbed samples from a mass spectrum to be analyzed so that the analysis is carried out without decreasing its accuracy.
Furthermore, the present inventors improve the analytic accuracy of the system by performing a sample measurement and a data analysis using the fact that multiply-charged ions with 5 or more charges when a genomic DNA sample is ionized by an ionization process using an air atomization.
Furthermore, the present inventors provide a genome DNA analysis system that allows a speedy backup when any emergency occurs in the system. Such a backup can be attained by comprising means for holding a standard sample, a plurality of measurement systems, display means for displaying the results of analysis, communication means to make a communication with a system administrator, and so on.
The above and the other objects of the present invention will be attained by the following configurations of the DNA analysis system.
(1) In the first aspect of the present invention, a DNA analysis system for analyzing DNA polymorphism, includes: ionization means for generating plural kinds of multiply-charged ions of a test DNA fragment, where each of them has five or more charges; mass spectrometric means for performing a mass spectrometry on the multiply-charged ion formed by the ionization means; analyzing-result prediction means that predicts a mass spectrum pattern from the mass spectrometric means in each of two cases, where one is that the test DNA fragment is polymorphic and the other is that the test DNA fragment is not polymorphic, based on both information about the test DNA fragment and information about a polymorphism point; comparative processing means for comparing a plurality of the mass spectrum patterns predicted by the analyzing-result prediction means with the analyzed results of the test DNA fragment analyzed by the mass spectrometric means to determine a nucleic acid base on the polymorphism point.
The information about the test DNA fragment includes the number of each of four different nucleic acid bases (i.e., adenine, thymine, guanine, and cytosine) that make up the test DNA fragment.
(2) In the DNA analysis system as set forth in the item (1), the analyzing-result prediction means predicts a mass-to-charge ratio (m/z; m is an ion mass, z is the number of electric charges) of the plural kinds of multi-charged ions in each of two cases, where one is that the test DNA fragment is polymorphic and the other is that the test DNA fragment is not polymorphic.
(3) In the DNA analysis system as set forth in the item (1), the analyzing-result prediction means predicts a mass-to-charge ratio (m/z; m is an ion mass, z is the number of electric charges) of the plural kinds of multi-charged ions and distribution of ion intensities in each of two cases, where one is that the test DNA fragment is polymorphic and the other is that the test DNA fragment is not polymorphic.
(4) In the DNA analysis system as set forth in the item (1), the DNA analysis system further includes: sampling means for supplying a sample including test DNA fragments to the ionization means at fixed intervals; and detecting-output analysis means for subtracting a mass spectrum obtained as an analyzing result with respect to a sample previously measured and modified by weight from a mass spectrum obtained as a detecting-output of the mass spectrometric means, wherein the mass spectrum processed by the detecting-output analysis means is provided as an analyzing result with respect of the test DNA fragment in the sample.
(5) In the DNA analysis system as set forth in the item (1), the ionization means generates multiply-charged ions of the test DNA fragment by the ionization means using an air atomization.
(6) In the DNA analysis system as set forth in the item (1), a nucleic acid base of a single nucleotide polymorphism point in the test DNA fragment is specified.
(7) In the DNA analysis system as set forth in the item (4), the DNA analysis system further includes: a display means for displaying an occurrence of an emergency when a maximum ion intensity detected by the mass spectrometric means is smaller than a predetermined threshold.
(8) In the DNA analysis system as set forth in the item (7), the DNA analysis system further includes: communication means for sending information about the occurrence of an emergency to a system administrator.
(9) In the DNA analysis system as set forth in the item (4), the sampling means introduces a standard sample into the ionization means when a maximum ion intensity of the mass spectrum detected by the mass spectrometric means is smaller than a predetermined threshold.
(10) In the DNA analysis system as set forth in the item (9), when a maximum ion intensity of a mass spectrum of the standard sample detected by the mass spectrometric means is equal to or higher than the threshold, the sample where the maximum ion intensity of the mass spectrum is detected as one smaller than the threshold is re-supplied to the ionization means by the sampling means.
(11) In the DNA analysis system as set forth in the item (9), the DNA analysis system further includes: a plurality of measurement systems, where each of the measurement systems comprises the sampling means, the ionization means, and the mass spectrometric means, when a maximum ion intensity of a mass spectrum of the standard sample detected by mass spectrometric means in one measurement system of the plurality of measurement systems is smaller than the threshold, the sample where a maximum ion intensity is detected as one smaller than the threshold at the one measurement system is transmitted to sampling means of another measurement system except the one measurement system.
(12) In the DNA analysis system as set forth in the item (9), the DNA analysis system further includes: a plurality of measurement systems, where each of the measurement systems comprises the sampling means, the ionization means, and the mass spectrometric means, wherein when a maximum ion intensity of a mass spectrum of the standard sample detected by mass spectrometric means in one measurement system of the plurality of measurement systems is smaller than the threshold, a sample intended to be measured by the one measurement system is sent to sampling means of another measurement system except the one measurement system.